Phosphotyrosine Protein Research Articles

Effects of oridonin on sperm function and the PI3K/PDK1/AKT signaling pathway: Implications for reproductive toxicity

Oridonin, a natural terpenoid isolated from the leaves of Isodon rubescens (Hemsley) H.Hara, is widely used in oriental medicine for its anticancer properties across various cancer types. Despite its prevalent use, the toxic effects of oridonin on male reproduction, particularly its impact on sperm functions and the mechanisms involved, are not well understood. This study aimed to explore the effects and underlying mechanisms of oridonin on sperm functions. We initially treated Duroc boar spermatozoa with varying concentrations of oridonin (0, 5, 50, 75, 100, and 150 µM) and incubated them to induce capacitation. We then assessed cell viability and several sperm functions, including sperm motility and motion kinematics, capacitation status, and ATP levels. We also analyzed the expression levels of proteins associated with the phosphatidylinositol 3-kinase (PI3K)/phosphoinositide-dependent kinase-1 (PDK1)/protein kinase B (AKT) signaling pathway and phosphotyrosine proteins. Our results indicate that oridonin adversely affects most sperm functions in a dose-dependent manner. We observed significant decreases in AKT, p-AKT (Thr308), phosphatase and tensin homolog (PTEN), p-PDK1, and p-PI3K levels following oridonin treatment, alongside an abnormal increase in phosphotyrosine proteins. These findings suggest that oridonin may disrupt normal levels of tyrosine-phosphorylated proteins by inhibiting the PI3K/PDK1/AKT signaling pathway, which is crucial for cell proliferation, metabolism, and apoptosis, thus potentially harming sperm functions. Consequently, we recommend considering the reproductive toxicity of oridonin when using it as a therapeutic agent.

METTL14-mediated N6-methyladenosine modification induces the ferroptosis of hypoxia/reoxygenation-induced cardiomyocytes

BackgroundHypoxia/reoxygenation (H/R) induces cardiomyocyte ferroptosis, a core remodeling event in myocardial ischemia/reperfusion injury. Methyltransferase-like 14 (METTL14) emerges as a writer of N6-methyladenosine (m6A) modification. This study was conducted to decipher the role of METTL14 in H/R-induced cardiomyocyte ferroptosis.MethodsMouse cardiomyocytes HL-1 were cultured and underwent H/R treatment. The degree of ferroptosis after H/R treatment was appraised by the cell counting kit-8 assay, assay kits (ROS/GSH/Fe2+), and Western blotting (GPX4/ACSL4). The intracellular expressions of METTL14, pri-miR-146a-5p, miR-146a-5p, or adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) were examined by real-time quantitative polymerase chain reaction or Western blotting, with m6A quantification analysis and RNA immunoprecipitation to determine the total m6A level and the expression of pri-miR-146a-5p bound to DiGeorge critical region 8 (DGCR8) and m6A-modified pri-miR-146a-5p. The binding of miR-146a-5p to APPL1 was testified by the dual-luciferase assay.ResultsH/R treatment induced cardiomyocyte ferroptosis (increased ROS, Fe2+, and ACSL4 and decreased GSH and GPX4) and upregulated METTL14 expression. METTL14 knockdown attenuated H/R-induced cardiomyocyte ferroptosis. METTL14 induced the recognition of pri-miR-146a-5p by DGCR8 by increasing m6A modification on pri-miR-146a-5p, which promoted the conversion of pri-miR-146a-5p into miR-146a-5p and further repressed APPL1 transcription. miR-146a-5p upregulation or APPL1 downregulation limited the inhibitory effect of METTL14 downregulation on H/R-induced cardiomyocyte ferroptosis.ConclusionMETTL14 promoted miR-146a-5p expression through the recognition and processing of pri-miR-146a-5p by DGCR8, which repressed APPL1 transcription and triggered H/R-induced cardiomyocyte ferroptosis.

Dibutyl phthalate disrupts [Ca2+]i, reactive oxygen species, [pH]i, protein kinases and mitochondrial activity, impairing sperm function

To explore the mechanism of sperm dysfunction caused by dibutyl phthalate (DBP), the effects of DBP on intracellular [Ca2+] and [pH], reactive oxygen species (ROS), lipid peroxidation (LPO), mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) levels, phosphorylation of protein kinase A (PKA) substrate proteins and phosphotyrosine (p-Tyr) proteins, sperm motility, spontaneous acrosome reaction, and tail bending were examined in mouse spermatozoa. At 100 µg/mL, DBP significantly increased tail bending and [Ca2+]i. Interestingly, DBP showed biphasic effects on [pH]i. DBP at 10–100 µg/mL significantly decreased sperm motility. Similarly, Ca2+ ionophore A23187 decreased [pH]i sperm motility, suggesting that DBP-induced excessive [Ca2+]i decreased sperm motility. DBP significantly increased ROS and LPO. DBP at 100 µg/mL significantly decreased mPTP closing, MMP, and ATP levels in spermatozoa, as did H2O2, indicative of ROS-mediated mitochondrial dysfunction caused by DBP. DBP as well as H2O2 increased p-Tyr sperm proteins and phosphorylated PKA substrate sperm proteins. DBP at 1–10 µg/mL significantly increased the spontaneous acrosome reaction, suggesting that DBP can activate sperm capacitation. Altogether, DBP showed a biphasic effect on intracellular signaling in spermatozoa. At concentrations relevant to seminal ortho-phthalate levels, DBP activates [pH]i, protein tyrosine kinases and PKA via physiological levels of ROS generation, potentiating sperm capacitation. DBP at high doses excessively raises [Ca2+]i and ROS and disrupts [pH]i, impairing the mitochondrial function, tail structural integrity, and sperm motility.

Ritonavir Has Reproductive Toxicity Depending on Disrupting PI3K/PDK1/AKT Signaling Pathway.

Ritonavir (RTV) is an antiviral and a component of COVID-19 treatments. Moreover, RTV demonstrates anti-cancer effects by suppressing AKT. However, RTV has cytotoxicity and suppresses sperm functions by altering AKT activity. Although abnormal AKT activity is known for causing detrimental effects on sperm functions, how RTV alters AKT signaling in spermatozoa remains unknown. Therefore, this study aimed to investigate reproductive toxicity of RTV in spermatozoa through phosphoinositide 3-kinase/phosphoinositide-dependent protein kinase-1/protein kinase B (PI3K/PDK1/AKT) signaling. Duroc spermatozoa were treated with various concentrations of RTV, and capacitation was induced. Sperm functions (sperm motility, motion kinematics, capacitation status, and cell viability) and expression levels of tyrosine-phosphorylated proteins and PI3K/PDK1/AKT pathway-related proteins were evaluated. In the results, RTV significantly suppressed sperm motility, motion kinematics, capacitation, acrosome reactions, and cell viability. Additionally, RTV significantly increased levels of phospho-tyrosine proteins and PI3K/PDK1/AKT pathway-related proteins except for AKT and PI3K. The expression level of AKT was not significantly altered and that of PI3K was significantly decreased. These results suggest RTV may suppress sperm functions by induced alterations of PI3K/PDK1/AKT pathway through abnormally increased tyrosine phosphorylation. Therefore, we suggest people who use or prescribe RTV need to consider its male reproductive toxicity.

Dual PTP1B/TC-PTP Inhibitors: Biological Evaluation of 3-(Hydroxymethyl)cinnoline-4(1H)-Ones

Dual inhibitors of protein phosphotyrosine phosphatase 1B (PTP1B)/T-cell protein phosphotyrosine phosphatase (TC-PTP) based on the 3-(hydroxymethyl)-4-oxo-1,4-dihydrocinnoline scaffold have been identified. Their dual affinity to both enzymes has been thoroughly corroborated by in silico modeling experiments. The compounds have been profiled in vivo for their effects on body weight and food intake in obese rats. Likewise, the effects of the compounds on glucose tolerance, insulin resistance, as well as insulin and leptin levels, have been evaluated. In addition, the effects on PTP1B, TC-PTP, and Src homology region 2 domain-containing phosphatase-1 (SHP1), as well as the insulin and leptin receptors gene expressions, have been assessed. In obese male Wistar rats, a five-day administration of all studied compounds led to a decrease in body weight and food intake, improved glucose tolerance, attenuated hyperinsulinemia, hyperleptinemia and insulin resistance, and also compensatory increased expression of the PTP1B and TC-PTP genes in the liver. The highest activity was demonstrated by 6-Chloro-3-(hydroxymethyl)cinnolin-4(1H)-one (compound 3) and 6-Bromo-3-(hydroxymethyl)cinnolin-4(1H)-one (compound 4) with mixed PTP1B/TC-PTP inhibitory activity. Taken together, these data shed light on the pharmacological implications of PTP1B/TC-PTP dual inhibition, and on the promise of using mixed PTP1B/TC-PTP inhibitors to correct metabolic disorders.

Regulation of Phosphorylation of Glycogen Synthase Kinase 3α and the Correlation with Sperm Motility in Human.

To unravel the mechanism regulating the phosphorylation of glycogen synthase kinase 3 (GSK3) and the correlation between the inhibitory phosphorylation of GSK3α and sperm motility in human. The phosphorylation and priming phosphorylated substrate-specific kinase activity of GSK3 were examined in human spermatozoa with various motility conditions. In human spermatozoa, GSK3α/β was localized in the head, midpiece, and principal piece of tail and p-GSK3α(Ser21) was enriched in the midpiece. The ratio of p-GSK3α(Ser21)/GSK3α was positively coupled with normal sperm motility criteria of World Health Organization. In high-motility spermatozoa, p-GSK3α(Ser21) phosphotyrosine (p-Tyr) proteins but p-GSK3α(Tyr279) markedly increased together with decreased kinase activity of GSK3 after incubation in Ca2+ containing medium. In high-motility spermatozoa, p-GSK3α(Ser21) levels were negatively coupled with kinase activity of GSK3, and which was deregulated in low-motility spermatozoa. In high-motility spermatozoa, 6-bromo-indirubin-3'-oxime, an inhibitor of kinase activity of GSK3 increased p-GSK3α(Ser21) and p-Tyr proteins. p-GSK3α(Ser21) and p-Tyr protein levels were decreased by inhibition of PKA and Akt. Calyculin A, a protein phosphatase-1/2A inhibitor, markedly increased the p-GSK3α(Ser21) and p-Tyr proteins, and significantly increased the motility of low-motility human spermatozoa. Down regulation of kinase activity of GSK3α by inhibitory phosphorylation was positively coupled with human sperm motility, and which was regulated by Ca2+, PKA, Akt, and PP1. Small-molecule inhibitors of GSK3 and PP1 can be considered to potentiate human sperm motility.

SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly.

CTL-mediated killing of virally infected or malignant cells is orchestrated at the immune synapse (IS). We hypothesized that SARS-CoV-2 may target lytic IS assembly to escape elimination. We show that human CD8+ T cells upregulate the expression of ACE2, the Spike receptor, during differentiation to CTLs. CTL preincubation with the Wuhan or Omicron Spike variants inhibits IS assembly and function, as shown by defective synaptic accumulation of TCRs and tyrosine phosphoproteins as well as defective centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing and cytokine production. These defects were reversed by anti-Spike antibodies interfering with ACE2 binding and reproduced by ACE2 engagement by angiotensin II or anti-ACE2 antibodies, but not by the ACE2 product Ang (1-7). IS defects were also observed ex vivo in CTLs from COVID-19 patients. These results highlight a new strategy of immune evasion by SARS-CoV-2 based on the Spike-dependent, ACE2-mediated targeting of the lytic IS to prevent elimination of infected cells.

Bombyxin-stimulated ecdysteroidogenesis in relation to sugar transporter/trehalase expressions in Bombyx prothoracic glands

Our previous studies showed that bombyxin stimulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs) during a long-term incubation period in a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner. In the present study, we further investigated the downstream signaling cascade in bombyxin-stimulated PGs. Our results showed that upon treatment with bombyxin, expression levels of the sugar transport 1 (St1) and St4 genes and trehalase 1 (Treh1) gene, but not ecdysteroid biosynthesis genes were greatly enhanced compared to the controls. Treatment with LY294002 (an inhibitor of PI3K) reduced the enhanced St1 and Treh1 expression levels, clearly indicating the involvement of PI3K. Treatment with 1 mM of mpV(pic) (a potent inhibitor of protein phosphotyrosine phosphatase and activator of insulin receptor (InR) kinase) also stimulated expression levels of the St1 and Treh1 genes, thus further confirming the involvement of the InR. Determining Treh enzyme activity showed that bombyxin treatment stimulated Treh enzyme activity in time- and PI3K-dependent manners. Validamycin A (a Treh inhibitor) blocked bombyxin-stimulated Treh enzyme activity and partly decreased bombyxin-stimulated ecdysteroidogenesis. A specific sugar transport inhibitor (cytochalasin B) and a glycolysis inhibitor (2-deoxy-D-glucose (2-DG)) also reduced bombyxin-stimulated ecdysteroidogenesis. Taken together, these results indicated that increased expressions of Sts and Treh1 and enhanced Treh enzyme activity downstream of InR/PI3K are involved in bombyxin-stimulated ecdysteroidogenesis in B. mori PGs.

Destabilization of the SHP2 and SHP1 protein tyrosine phosphatase domains by a non-conserved “backdoor” cysteine

Protein tyrosine phosphatases (PTPs) are critical regulators of cellular signal transduction that catalyze the hydrolytic dephosphorylation of phosphotyrosine in substrate proteins. Among several conserved features in classical PTP domains are an active-site cysteine residue that is necessary for catalysis and a “backdoor” cysteine residue that can serve to protect the active-site cysteine from irreversible oxidation. Curiously, two biologically important phosphatases, Src homology domain-containing PTPs 2 and 1 (SHP2 and SHP1), each contain an additional backdoor cysteine residue at a position of the PTP domain that is occupied by proline in almost all other classical PTPs (position 333 in human SHP2 numbering). Here we show that the presence of cysteine 333 significantly destabilizes the fold of the PTP domains in the SHPs. We find that replacement of cysteine 333 with proline confers increased thermal stability on the SHP2 and SHP1 PTP domains, as measured by temperature-dependent activity assays and differential scanning fluorimetry. Conversely, we show that substantial destabilization of the PTP-domain fold is conferred by introduction of a non-natural cysteine residue in a non-SHP PTP that contains proline at the 333 position. It has previously been suggested that the extra backdoor cysteine of the SHP PTPs may work in tandem with the conserved backdoor cysteine to provide protection from irreversible oxidative enzyme inactivation. If so, our current results suggest that, during the course of mammalian evolution, the SHP proteins have developed extra protection from oxidation at the cost of the thermal instability that is conferred by the presence of their PTP domains’ second backdoor cysteine.

Reproductive toxicity of ritonavir in male: Insight into mouse sperm capacitation

Since COVID-19 began in 2019, therapeutic agents are being developed for its treatment. Among the numerous potential therapeutic agents, ritonavir (RTV), an anti-viral agent, has recently been identified as an important element of the COVID-19 treatment. Moreover, RTV has also been applied in the drug repurposing of cancer cells. However, previous studies have shown that RTV has toxic effects on various cell types. In addition, RTV regulates AKT phosphorylation within cancer cells, and AKT is known to control sperm functions (motility, capacitation, and so on). Although deleterious effects of RTV have been reported, it is not known whether RTV has male reproduction toxicity. Therefore, in this study, we aimed to investigate the effects of RTV on sperm function and male fertility. In the present study, sperm collected from the cauda epididymis of mice were incubated with various concentrations of RTV (0, 0.1, 1, 10, and 100 μM). The expression levels of AKT, phospho-AKT (Thr308 and Ser473), and phospho-tyrosine proteins, sperm motility, motion kinematics, capacitation status, and cell viability were assessed after capacitation. The results revealed that AKT phosphorylation at Thr308 and Ser473 was significantly increased, and the levels of tyrosine-phosphorylated proteins (at approximately 25 and 100 kDa) were significantly increased in a dose-dependent manner. In addition, RTV adversely affected sperm motility, motion kinematics, and cell viability. Taken together, RTV may have negative effects on sperm function through an abnormal increase in tyrosine phosphorylation and phospho-AKT levels. Therefore, individuals taking or prescribing RTV should be aware of its reproductive toxicity.

Glycogen Synthase Kinase-3 Isoform Variants and Their Inhibitory Phosphorylation in Human Testes and Spermatozoa.

To clarify (phospho-) glycogen synthase kinase-3 (GSK3) isoform variants in the germline and soma of human testes and spermatozoa. GSK3 isoform variants in normospermatogenic and Sertoli cell-only (SCO) testicular biopsies and spermatozoa were examined. In normospermatogenic testes, GSK3α and GSK3β variants 1 and 2 different in low complexity region (LCR) were expressed and their levels were decreased in SCO testes. GSK3β variant 3 was only expressed in SCO testes. GSK3β as well as GSK3α, the dominant isoforms in testes were decreased in SCO testes. In normospermatogenic testes, GSK3β were found in spermatogonia and markedly decreased in meiotic germ cells in which GSK3α was dominant. p-GSK3α/β were marginal in spermatogonia and early spermatocytes. In SCO testes, GSK3α/β immunoreactivity in seminiferous epithelia was weaker than those of normospermatogenic testes whereas p-GSK3α/β(Ser) immunoreactivity was visibly increased in Sertoli cells. GSK3α was dominant in ejaculated spermatozoa in which GSK3α and p-GSK3α(Ser) were found in the head, midpiece, and tail. In acrosome-reacted spermatozoa, GSK3α was found in the equatorial region of head, midpiece, and tail, and p-GSK3α(Ser) was only found in midpiece. During sperm capacitation, p-GSK3α(Ser) was significantly increased together with phosphotyrosine proteins and motility. In human male germ cells, GSK3 isoforms different in LCRs switch from GSK3β to GSK3α during meiotic entry, suggesting the isoform-specific roles of GSK3α and GSK3β in meiosis and stemness or proliferation of spermatogonia, respectively. In dormant Sertoli cells of SCO testes kinase activity of GSK3 might be downregulated via inhibitory phosphorylation. In spermatozoa, inhibitory phosphorylation of GSK3α might be coupled with activation of motility during capacitation.

Characterization and biological activity of new 4-oxo-1,4-dihydrocinnoline-based inhibitors of the tyrosine phosphatase PTP1B and TCPTP

Functional disorders in obesity are largely due to a decrease in tissue sensitivity to insulin and leptin. One of the ways to restore it is inhibition of protein phosphotyrosine phosphatase 1B (PTP1B) and T-cell protein phosphotyrosine phosphatase (TCPTP), negative regulators of the insulin and leptin signaling. Despite progress in the development of inhibitors of these phosphatases, commercial preparations based on them have not been developed yet, and the mechanisms of action are poorly understood. The aim of the work was to study the effect of new derivatives of 4-oxo-1,4-dihydrocinnoline (PI04, PI06, PI07) on the activity of PTP1B and TCPTP, as well as to study the effect of their five-day administration (i.p., 10 mg/kg/day) to Wistar rats with diet-induced obesity on body weight and fat, metabolic and hormonal parameters, and gene expression of phosphatase and insulin and leptin receptors in the liver. It has been shown that PI04 is a mild, low selective inhibitor of both phosphatases (PTP1B, IC50=3.42(2.60-4.51) μM; TCPTP, IC50=4.16(3.49-4.95) μM), while PI06 and PI07 preferentially inhibit PTP1B (IC50=3.55 (2.63-4.78) μM) and TCPTP (IC50=1.45(1.18-1.78) μM), respectively. PI04 significantly reduced food intake, body weight and fat, attenuated hyperglycemia, normalized glucose tolerance, basal and glucose-stimulated levels of insulin and leptin, and insulin resistance index. Despite the anorexigenic effect, PI06 and PI07 were less effective, having little effect on glucose homeostasis and insulin sensitivity. PI04 significantly increased the expression of the PTP1B and TCPTP genes and decreased the expression of the insulin and leptin receptor genes. PI06 and PI07 had little effect on these indicators. Thus, PI04, the inhibitor of PTP1B and TCPTP phosphatases, restored metabolic and hormonal parameters in obese rats with greater efficiency than inhibitors of PTP1B (PI06) and TCPTP (PI07). This indicates the prospect of creating mixed PTP1B/TCPTP inhibitors for correction of metabolic disorders.

APPL1 ameliorates myocardial ischemia-reperfusion injury by regulating the AMPK signaling pathway.

Myocardial ischemia-reperfusion injury results in elevated reactive oxygen species (ROS) production and causes oxidative stress damage. Therefore, the current study aimed to investigate whether adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) could induce the expression of antioxidant enzymes through AMP-activated protein kinase (AMPK) signaling in order to alleviate the injury caused by ischemia/hypoxia-reperfusion. Following induction of hypoxia-reoxygenation (H/R) injury in H9c2 cells, the liver kinase B1 (LKB1)/AMPK/acetyl-CoA carboxylase α (ACC) signaling pathway was investigated using western blot analysis, along with the detection of superoxide dismutase (SOD)2 and SOD3 expression. Additionally, cell viability was detected using a Cell Counting Kit-8 assay and ROS production was analyzed using ROS staining, whereas the expression levels of inflammatory mediators (TNF-α, monocyte chemoattractant protein 1 and IL-1β), apoptosis mediators [cleaved caspase-3, cleaved poly (ADP-ribose) polymerase and Bcl-2] and nuclear factor erythroid 2-related factor 2 signaling pathway-related proteins were detected via western blot analysis following overexpression of APPL1 alone or in combination with compound C treatment (an AMPK inhibitor). The results indicated that H/R induction upregulated the phosphorylation levels of LKB1, AMPK and ACC, and decreased the expression levels of APPL1 and SOD enzyme activities. APPL1 overexpression increased the phosphorylation levels of LKB1, AMPK and ACC, SOD enzyme activity and cell viability whereas the expression levels of proinflammatory mediators and proapoptotic mediators, and the levels of ROS production were markedly decreased when compared with H/R group with empty plasmid transfection. APPL overexpression-mediated effects were significantly abrogated by compound C. Taken together, the data indicated that APPL1 inhibited ROS production and H/R-induced myocardial injury via the AMPK signaling pathway. Therefore, APPL1 may serve as a potential therapeutic target for myocardial H/R injury.

Adiponectin inhibits D‑gal‑induced cardiomyocyte senescence via AdipoR1/APPL1.

The aim of the present study was to examine whether adiponectin could inhibit cardiomyocyte senescence induced by D-galactose (D-gal), and whether it functioned via the adiponectin receptor 1 (AdipoR1)/adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) signaling pathway. For this purpose, the expression levels of adiponectin, AdipoR1 and APPL1 in mouse plasma and myocardial tissues were detected via reverse transcription-quantitative PCR (RT-qPCR) and western blotting. An adiponectin-overexpression plasmid was transfected into D-gal-treated H9c2 cells prior to the detection of AdipoR1 and APPL1 expression by RT-qPCR. Senescence-associated β-galactose staining was then performed to observe cellular senescence following the transfection of small interfering RNAs (si) targeting AdipoR1 and APPL1 into D-gal-treated H9c2 cells overexpressing adiponectin. Commercial kits were used to detect reactive oxygen species (ROS) production and malondialdehyde (MDA) content in the different groups. The expression levels of heme oxygenase (HO)-1 and high mobility group box 1 (HMGB1) were examined by western blot analysis. The results revealed that the expression levels of adiponectin, AdipoR1 and APPL1 were downregulated in aged mouse plasma, myocardial tissues and D-gal-treated cardiomyocytes. It was also observed that AdipoR1 and APPL1 expression levels were significantly upregulated following the overexpression of adiponectin into D-gal-treated cardiomyocytes. Moreover, adiponectin overexpression reduced cellular senescence induced by D-gal and the expression of p16 and p21; these effects were reversed following transfection with si-AdipoR1 and si-APPL1. Adiponectin also downregulated the levels of ROS and MDA in D-gal-treated H9c2 cells via AdipoR1/APPL1. Additionally, the release of HO-11/HMGB1 was affected by adiponectin via AdipoR1/APPL1, and adiponectin/AdipoR1/APPL1 suppressed ROS production via HO-1/HMGB1. On the whole, the present study demonstrated that adiponectin played an inhibitory role in cardiomyocyte senescence via the AdioR1/APPL1 signaling pathway and inhibited the levels of oxidative stress in senescent cardiomyocytes via the HO-1/HMGB1 signaling pathway.

Distinct Proteomic Profiling of Plasma Extracellular Vesicles from Moderate-to-Severe Atopic Dermatitis Patients.

BackgroundAtopic dermatitis (AD) is a chronic, inflammatory cutaneous disorder characterized by a T helper 2 (Th2) immune response phenotype. Extracellular vesicles (EVs) are a heterogeneous family of cell-derived membranous structures, which transport cellular components such as DNA and proteins, and are involved in multiple physiological and pathological processes. Increasing evidence has shown that EVs secretion took part in the pathogenesis of AD. However, the proteomic studies of plasma-derived EVs in AD patients have not been reported.ObjectiveIn this study, we investigated the diversity of plasma EVs collected from AD patients and healthy individuals and suggested that the candidates for uniquely or differentially expressed proteins in plasma EVs could be a diagnostic marker in AD.MethodsThe plasma EVs were collected from 12 patients with moderate-to-severe AD and 13 healthy subjects. Proteomic analysis was performed by using a comprehensive nanoLC‑MS/MS method.ResultsProteomic analysis revealed that a total of 1478 proteins in plasma EVs were found to be common proteins in AD, whereas a total of 1597 proteins in plasma EVs were found to be common proteins in HC. Eighty-six proteins in plasma EVs showed more than 2.5-fold up-regulation, while a total of 225 proteins in plasma EVs showed less than 1/2.5-fold down-regulation with a significant difference (p < 0.05) among AD compared with HC. The candidates for differentially expressed proteins in plasma EVs have been described as a connectivity PPI network related to several KEGG pathways, including pathways in platelet activation, complement, and so on.ConclusionSLP-76 tyrosine phosphoprotein (SLP76) involved in platelet activation may significantly contribute to the pathogenesis of AD. We will further verify the role of SLP67 in AD via animal and cell experiments to provide a promising therapeutic or diagnostic target.

Increasing LFA-1 Expression Enhances Immune Synapse Architecture and T Cell Receptor Signaling in Jurkat E6.1 Cells.

The Jurkat E6.1 clone has been extensively used as a powerful tool for the genetic and biochemical dissection of the TCR signaling pathway. More recently, these cells have been exploited in imaging studies to identify key players in immunological synapse (IS) assembly in superantigen-specific conjugates and to track the dynamics of signaling molecules on glass surfaces coated with activating anti-CD3 antibodies. By comparison, Jurkat cells have been used only scantily for imaging on supported lipid bilayers (SLBs) incorporating laterally mobile TCR and integrin ligands, which allow to study synaptic rearrangements of surface molecules and the fine architecture of the mature IS, likely due to limitations in the assembly of immune synapses with well-defined architecture. Here we have explored whether upregulating the low levels of endogenous LFA-1 expression on Jurkat E6.1 cells through transduction with CD11a- and CD18-encoding lentiviruses can improve IS architecture. We show that, while forced LFA-1 expression did not affect TCR recruitment to the IS, E6.1 LFA-1high cells assembled better structured synapses, with a tighter distribution of signaling-competent TCRs at the center of the IS. LFA-1 upregulation enhanced protein phosphotyrosine signaling on SLBs but not at the IS formed in conjugates with SEE-pulsed APCs, and led to the constitutive formation of an intracellular phosphotyrosine pool co-localizing with endosomal CD3ζ. This was paralleled by an increase in the levels of p-ZAP-70 and p-Erk both under basal conditions and following activation, and in enhanced Ca2+ mobilization from intracellular stores. The enhancement in early signaling E6.1 LFA-1high cells did not affect expression of the early activation marker CD69 but led to an increase in IL-2 expression. Our results highlight a new role for LFA-1 in the core architecture of the IS that can be exploited to study the spatiotemporal redistribution of surface receptors on SLBs, thereby extending the potential of E6.1 cells and their derivatives for fine-scale imaging studies.

Integrated Phosphoproteomic and N-Glycoproteomic Landscape of Cutaneous T-Cell Lymphoma Reveals Distinctive Pathogenetic Insights and Therapeutic Vulnerabilities

Background: A critical goal of precision oncology is the identification of deregulated signaling pathways that serve as disease-specific biomarkers and vulnerabilities that may be exploited as therapeutic targets. Cutaneous T cell lymphomas (CTCLs) are heterogeneous neoplasms with variable clinical outcomes. Whereas the genomic landscapes of CTCLs are being characterized, a large scale analysis of their proteomic signatures and post-translational modifications (PTM) has not been performed. In this study, we sought to gain insights into the pathogenesis of CTCLs using high mass accuracy tandem mass spectrometry (HMA-MS/MS)-based phosphoproteomics and glycoproteomics.Methods: CTCL-derived cell lines representative of Sézary syndrome (SS, HUT78), mycosis fungoides (MF, HH), and ALK-negative anaplastic large cell lymphoma (ALCL) with PCM1-JAK2 or NPM1-TYK2 translocations (Mac1, Mac2a, and Myla) were analyzed in technical and biologic triplicates to perform unbiased proteome characterization. Phosphoproteins were enriched using metal oxide affinity chromatography and immunoprecipitation using 3 anti-phosphotyrosine antibodies and N-glycoproteins were enriched using solid phase extraction and hydrazide resin and an Orbitrap Fusion or LTQ OrbitrapXL in-line with a Paradigm MS2 HPLC. Precise site-mapping of the PTMs was assigned and quantitated using Maxquant. Hierarchical clustering was performed using Perseus. Whole exome sequencing (WES) was performed using an Illumina HiSeq 2500. Functional studies using tyrosine kinase inhibitors were performed to assess selective vulnerabilities.Results: Phosphoproteomic analysis identified 1899 tyrosine (Y) phosphoproteins and 6029 serine/threonine (S/T) phosphoproteins. Unsupervised hierarchical clustering of the Y-phosphoproteome data accurately grouped the CTCLs to those with and without underlying tyrosine kinase translocations. Unique driver kinase phosphosignaling signatures were identified in MYLA (NPM1-TYK2) and MAC1/2 (PCM1-JAK2) representing the ALCL subgroup, as well as close similarity between the signatures of MF and SS-derived cells (HH and HUT78). Notably, phosphoproteomic analysis of HUT78 (SS) yielded high spectral counts of Y-phosphorylated residues at IL2RG p.Y325, JAK3 p.Y980/Y981, and STAT5 p.Y699 indicative of constitutive activation of the JAK/STAT5 pathway corroborated by WES showing activating mutations in JAK3 p.A573V and JAK1 p.Y654F.Beyond driver kinases, oncogenic kinase-specific substrates with distinct site-specific phosphorylated residues were identified for each disease class. Phosphoproteins selectively expressed in MF/SS, (46 p-S/T and 25 p-Y proteins) included members of the killer-cell immunoglobulin-like receptor (KIR) family implicated in immune modulation through immune tyrosine-based inhibitor or TYRO protein tyrosine kinase binding domains for signal transduction. Additionally, proteins not previously associated with MF/SS including NKG2-D type integral membrane protein family members were discriminative of this subgroup. Phosphoprotein members of lysine demethylases (KDM family) and Aryl Hydrocarbon Receptor families were distinctively identified in MF/SS cells, while components of the T-cell receptor signaling pathways were common in all groups of CTCLs. By comparison, N-glycoproteomic analysis identified 811 unique N-glycoproteins. A total of 51 N-glycoproteins were found solely in HUT78 of which 46 were novel in their specificity and included 13 with a role in cell-adhesion while N-glycoproteins unique to HH revealed 12 novel candidate biomarkers (e.g. MERTK, CD97 and SLC44A2). Integrative phosphoproteomic and N-glycoproteomic analysis strikingly revealed convergent pathways implicating a prominent role for the KIR family proteins in SS. Orthogonal immunophenotyping by flow cytometry has shown high sensitivity (96%) detection of KIR3DL2 in primary human SS cases. Significantly, anti-KIR3DL2 inhibition using a humanized antibody revealed activity against SS cells. By comparison, TYK2-driven CTCL (MYLA) was sensitive to inhibitors of TYK2 and JAK.Conclusions: Integrated phosphoproteomic and N-glycoproteomic analysis identifies known and novel candidate biomarkers, unique PTM signatures, and therapeutic vulnerabilities implicating disease-specific pathogenetic mechanisms in CTCL. DisclosuresNo relevant conflicts of interest to declare.

GPVI Intracellular Signaling Is Activated in Platelets from Patients with Essential Thrombocythemia

GPVI Intracellular Signaling Is Activated in Platelets from Patients with Essential Thrombocythemia

ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model.

In boar sperm, we have previously shown that capacitation is associated with the appearance of the p32 tyrosine phosphoprotein complex. The principal tyrosine phosphoprotein involved in this complex is the acrosin-binding protein (ACRBP), which regulates the autoconversion of proacrosin to intermediate forms of acrosin in both boar and mouse sperm. However, the complete biological role of ACRBP has not yet been elucidated. In this study, we tested the hypothesis that tyrosine phophorylation and the presence of the ACRBP in the sperm head are largely necessary to induce capacitation, the acrosome reaction (AR) and sperm-zona pellucida (ZP) binding, all of which are necessary steps for fertilization. In vitro fertilization (IVF) was performed using matured porcine oocytes and pre-capacitated boar sperm cultured with anti-phosphotyrosine antibodies or antibodies against ACRBP. Anti-ACRBP antibodies reduced capacitation and spontaneous AR (P<0.05). Sperm-ZP binding declined in the presence of anti-phosphotyrosine or anti-ACRBP antibodies. The localisation of anti-ACRBP antibodies on the sperm head, reduced the ability of the sperm to undergo the AR in response to solubilized ZP or by inhibiting the sarco/endoplasmic reticulum Ca2+-ATPase. These results support our hypothesis that tyrosine phosphorylated proteins and ACRBP are present upon the sperm surface in order to participate in sperm-ZP binding, and that ACRBP upon the surface of the sperm head facilitates capacitation and the AR in the porcine.

Ouabain-induced activation of phospholipase C zeta and its contributions to bovine sperm capacitation.

The sperm-derived oocyte activating factor, phospholipase C zeta (PLC ζ), is the only PLC isoform reported in cattle. The objectives were to (1) localize PLC ζ in fresh and capacitated bovine sperm and (2) investigate the activation of PLC ζ during bull sperm capacitation and contributions of PLC activity to this process. We confirmed interaction of testis-specific isoform of Na/K-ATPase (ATP1A4) with PLC ζ (immunolocalization and immunoprecipitation) and tyrosine phosphorylation (immunoprecipitation) of PLC ζ (a post-translational protein modification commonly involved in activation of PLC in somatic cells) during capacitation. Furthermore, incubation of sperm under capacitating conditions upregulated PLC-mediated hyperactivated motility, tyrosine phosphoprotein content, acrosome reaction, and F-actin formation (flow cytometry), implying that PLC activity is enhanced during capacitation and contributing to these capacitation processes. In conclusion, we inferred that PLC ζ is activated during capacitation by tyrosine phosphorylation through a mechanism involving ATP1A4, contributing to capacitation-associated biochemical events.